MOST: detecting cancer differential gene expression

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1 Biostatistics (2008), 9, 3, pp doi: /biostatistics/kxm042 Advance Access publication on November 29, 2007 MOST: detecting cancer differential gene expression HENG LIAN Division of Mathematical Sciences, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore SUMMARY We propose a new statistics for the detection of differentially expressed genes when the genes are activated only in a subset of the samples. Statistics designed for this unconventional circumstance has proved to be valuable for most cancer studies, where oncogenes are activated for a small number of disease samples. Previous efforts made in this direction include cancer outlier profile analysis (Tomlins and others, 2005), outlier sum (Tibshirani and Hastie, 2007), and outlier robust t-statistics (Wu, 2007). We propose a new statistics called maximum ordered subset t-statistics (MOST) which seems to be natural when the number of activated samples is unknown. We compare MOST to other statistics and find that the proposed method often has more power then its competitors. Keywords: Cancer; COPA; Differential gene expression; Microarray. 1. INTRODUCTION The most popular method for differential gene expression detection in 2-sample microarray studies is to compute the t-statistics. The differentially expressed genes are those whose t-statistics exceed a certain threshold. Recently, many researchers have come to the realization that in many cancer studies, many genes show increased expressions in disease samples, but only for a small number of those samples. The study of Tomlins and others (2005) shows that t-statistics has low power in this case, and they introduced the so-called cancer outlier profile analysis (COPA). Their study shows clearly that COPA can perform better than the traditional t-statistics for cancer microarray data sets. More recently, several progresses have been made in this direction with the aim to design better statistics to account for the heterogeneous activation pattern of the cancer genes. In Tibshirani and Hastie (2007), the authors introduced a new statistics, which they called outlier sum. Later, Wu (2007) proposed outlier robust t-statistics (ORT) and showed it usually outperformed the previously proposed ones in both simulation study and application to real data set. In this paper, we propose another statistics for the detection of cancer differential gene expression which have similar power to ORT when the number of activated samples is very small, but perform better when more samples are differentially expressed. We call our new method the maximum ordered subset t-statistics (MOST). Through simulation studies, we found the new statistics outperformed the previously To whom correspondence should be addressed. c The Author Published by Oxford University Press. All rights reserved. For permissions, please journals.permissions@oxfordjournals.org.

2 412 H. LIAN proposed ones under some circumstances and never significantly worse in all situations. Thus, we think it is a valuable addition to the dictionary of cancer outlier expression detection. 2. MAXIMUM ORDERED SUBSET t-statistics We consider the simple 2-class microarray data for detecting cancer genes. We assume there are n normal samples and m cancer samples. The gene expressions for normal samples are denoted by x ij for genes i = 1, 2,...,p and samples j = 1, 2,...n, while y ij denote the expressions for cancer samples with i = 1, 2,...,p and j = 1, 2,...m. In this paper, we are only interested in 1-sided test where the activated genes from cancer samples have a higher expression level. The extension to 2-sided test is straightforward. The usual t-statistics (up to a multiplication factor independent of genes) for 2-sample test of differences in means is defined for each gene i by T i = x i ȳ i, (2.1) s i where x i = j x ij/n is the average expression of gene i in normal samples, ȳ i = j y ij/m is the average expression of gene i in cancer samples, and s i is the usual pooled standard deviation estimate 1 si 2 j n = (x ij x i ) j m (y ij ȳ i ) 2. n + m 2 The t-statistics is powerful when the alternative distribution is such that y ij, j = 1, 2,...,m, all come from a distribution with a higher mean. Tomlins and others (2005) argues that for most cancer types, heterogeneous activation patterns make t-statistics inefficient for detecting those expression profiles. They defined the COPA statistics C i = q r ({y ij } 1 j m ) med i, (2.2) mad i where q r ( ) is the rth percentile of the data, med i = median({x ij } 1 j n, {y ij } 1 j m ) is the median of the pooled samples for gene i, and mad i = median({x ij med i } 1 j n, {y ij med i } 1 j m ) is the median absolute deviation of the pooled samples. The choice of r in (2.2) depends on the subjective judgement of the user. The use of med i and mad i to replace the mean and the standard deviation in (2.1) is due to robustness considerations since it is already known that some of the genes are differentially expressed. In (2.2), only one value of {y ij } is used in the computation. A more efficient strategy would be to use additional expression values. Let O i ={y ij : y ij > q 75 ({x ij } 1 j n, {y ij } 1 j m ) + IQR({x ij } 1 j n, {y ij } 1 j m )} (2.3) be the outliers from the cancer samples for gene i, where IQR( ) is the interquartile range of the data. The OS statistics from Tibshirani and Hastie (2007) is then defined as y ij O i (y ij med i ) OS i =. (2.4) mad i More recently, Wu (2007) studied ORT statistics, which is similar to OS statistics. The important difference that makes ORT superior is that outliers are defined relative to the normal sample instead of the pooled sample. So in their definition, O i ={y ij : y ij > q 75 ({x ij } 1 j n ) + IQR({x ij } 1 j n )}. (2.5)

3 MOST: detecting cancer differential gene expression 413 By similar reasoning, med i in OS is replaced by med ix and mad j by median({x ij med ix } 1 j n, {y ij med iy } 1 j m ), where med ix and med iy are the medians of normal and cancer samples, respectively. In both OS and ORT statistics, the outliers are defined somewhat arbitrarily with no convincing reasons. To address this question, we propose the following statistics that implicitly considers all possible values for outlier thresholds. Suppose for notational simplicity that {y ij } 1 j m are ordered for each i: y i1 y i2 y im. If the number of samples where oncogenes are activated is known, we would naturally define the statistics as 1 j k M ik = (y ij med ix ) median({x ij med ix } 1 j n, {y ij med iy } 1 j m ). (2.6) When k is not known to us, one would be tempted to define M i = max M ik. 1 k m But this does not quite work since obviously M ik for different values of k are not directly comparable under the null distribution that x ij, y ij N(0, 1). For example, when m = 2, we have E[y i1 med ix ] > 0, while E [ j=1,2 (y ij med ix ) ] = 0. This observation motivates us to normalize M ik such that each approximately has mean 0 and variance 1. This can be achieved by defining µ k = E [ 1 j k z j] and σk 2 = Var( 1 j k z j), where z1 > z 2 > > z m is the order statistics of m samples generated from the standard normal distribution. Then, we can define M ik as ( 1 j k M ik = (y )/ ij med ix ) median({x ij med ix } 1 j n, {y ij med iy } 1 j m ) µ k σ k (2.7) so that M ik has mean and variance approximately equal to 0 and 1, respectively. Finally, we can define our new statistics (called MOST) as M i = max M ik. (2.8) 1 k m With MOST, we practically consider every possible threshold above which y ij are taken to be outliers. In this formulation, the number of outliers is implicitly defined as arg max M ik. (2.9) 1 k m 3. SIMULATION STUDIES AND APPLICATION Some simulations are carried out to study MOST and compare its performance to OS, ORT, COPA, and t-statistics. For COPA, we choose to use the 90th percentile in its definition as in Tibshirani and Hastie (2007). We generate the expression data from standard normal with n = m = 20. For various values k, 1 k m, which is the number of differentially expressed cancer samples, a constant µ is added for differentially expressed genes. We simulated 1000 differentially and nondifferentially expressed genes and calculated the receiver operating characteristic (ROC) curves from them by choosing different thresholds for gene calls. Figures 1 and 2 plot the ROC curves for some combinations of k and µ. Forµ = 2 and k small, all 5 statistics behave similarly with t-statistics performing the worst. As k increases, t becomes better and OS and COPA begin to lose power. For µ = 1 and medium to large k, the performance of MOST is only worse than t and better than other statistics. Smaller k in this case basically leads to ROC curve that is close to a 45 line for all statistics since the signal µ = 1 is too weak in this case, so we do not show these

4 414 H. LIAN Fig. 1. ROC curves estimated based on simulation. The number of normal/cancer sample is n = m = 20. Various combinations of µ and k s are chosen. Other uninteresting results where all statistics have close to perfectly good or bad performances are excluded as explained in the main text.

5 MOST: detecting cancer differential gene expression 415 Fig. 2. More ROC curves. results. For µ = 4 and small k, MOST is better than ORT, COPA, and t, and in this situation, only OS is competitive with MOST. Larger k in this case will produce nearly perfect ROC curves for all statistics, and thus those results are also omitted. Besides ROC curves, we have also tried examining the possibility of using (2.9) for estimating the number of differentially expressed samples k but so far have been unable to get a reasonable estimate out of it. From the above simulations, we judge that our new estimate MOST is at least as good as other previously proposed statistics, sometimes much better. Thus, it is a valuable tool for detecting activated genes in many situations. As an example of real data application, the data from West and others (2001) is publicly available from The microarray used in the breast cancer study contains 7129 genes and 49 tumor samples, 25 of which with no positive lymph nodes identified and the other 24 with positive nodes. Similar to Wu (2007), we take the log transformation of the expressions after normalizing the data. We apply all the above mentioned statistics to the data. For real data application, we need to use 2-sided test. The modification for MOST required is straightforward. We just use 1 j k m ik = (y ij med ix ) median({x ij med ix } 1 j n, {y ij med iy } 1 j m ). (3.1)

6 416 H. LIAN This time with y ij ordered such that y i1 y i2 y im. We also need to normalize m ik as in (2.7) and then m i = min 1 k m m ik. This test will detect downward-regulated genes in disease samples. The 2-sided test will be the maximum of the absolute values of M i and m i, but the sign is kept. A search of PubMed returns 908 genes related to breast cancer, and these are mapped to 655 probe sets on the Affymetrix HuGeneFL microarray used in this experiment. The ranking of these are computed for all the 5 statistics. The differences in ranking between MOST and other statistics are plotted in Figure 3 as histograms. Negative values in the histograms show the superiority of the MOST statistics since they imply higher ranking to these breast cancer related genes given by MOST. The histograms show that for this data, the performance of MOST is superior to t-statistics (shown by the heavy left tail in the histogram) and similar to others. In a second example, we consider a subset of acute lymphoblastic leukemia (ALL) data consisting 79 samples patients with B-cell acute lymphoblastic leukemia, available from the Bioconductor project. The comparison is between 37 samples with the BCR/ABL fusion gene resulting from a translocation and 42 normal samples. In total, 358 probe sets on the array are annotated with the gene ontology term tyrosine Fig. 3. Comparison between MOST and other statistics on breast cancer data. The histogram shows the difference in ranking on 655 probe sets. In these figures, left-skewed histogram implies superiority of MOST.

7 MOST: detecting cancer differential gene expression 417 Fig. 4. Comparison between MOST and other statistics on ALL data. kinase activity, which is believed to mediate many of the effects due to BCR/ABL translocation. After a simple filtering to remove probe sets that are not expressed, 94 of those 358 probe sets remain. The differences in ranking between MOST and other statistics are shown as histograms in Figure 4. For this data, t-test and MOST seem to be superior to other methods. The R implementation of MOST statistics as well as sample code for simulation is available at REFERENCES TIBSHIRANI, R. AND HASTIE, T. (2007). Outlier sums for differential gene expression analysis. Biostatistics 8, 2 8. TOMLINS, S. A., RHODES, D. R., PERNER, S., DHANASEKARAN, S. M., MEHRA, R., SUN, X. W., VARAMBALLY, S., CAO, X., TCHINDA, J., KUEFER, R. and others (2005). Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science 310, WEST, M.,BLANCHETTE, C., DRESSMAN, H.,HUANG, E.,ISHIDA, S., SPANG, R., ZUZAN, H.,OLSON, J.A.J., MARKS, J. R. AND NEVINS, J. R. (2001). Predicting the clinical status of human breast cancer by using

8 418 H. LIAN gene expression profiles. Proceedings of the National Academy of Sciences of the United States of America 98, WU, B. (2007). Cancer outlier differential gene expression detection. Biostatistics 8, [Received August 8, 2007; first revision September 24, 2007; second revision October 23, 2007; accepted for publication October 25, 2007]